Periodontitis is a bacteria-driven inflammatory bone loss disease affecting 47% of adults in the United States. Oral pathogens, such as Aggregatibacter actinomycetemcomitans (Aa), the pathogen associated with localized aggressive periodontitis, stimulate mammalian cells to generate and release sphingosine-1-phosphate (S1P). S1P binds to five G protein-coupled receptors, which initiates various cellular signaling pathways and affects many physiological and pathophysiological processes. However, it is unknown how S1P signaling modulates the inflammatory bone loss response induced by oral pathogens. The long-term goal is to understand the role of S1P signaling in regulating the immune response to oral pathogens and to develop a novel therapeutic strategy for periodontitis. Our preliminary study demonstrated that S1P signaling is critical in regulating the immune response to Aa. In a periodontal inflammatory bone loss animal model, deficiency in generation of S1P in mice (sphingosine kinase 1 KO mice) attenuated periodontal leukocyte infiltration and alleviated alveolar bone loss in response to Aa stimulation. Additionally, pharmacological inhibition or RNA silencing of S1P receptor 2 (S1PR2) in murine bone marrow-derived macrophages (BMM) significantly attenuated COX-2, IL-1, IL-6, and TNF mRNA expressions induced by Aa. Furthermore, pharmacological inhibition of S1PR2 in BM-derived preosteoclasts suppressed osteoclastogenesis induced by Aa-stimulated conditioned media. The overall objective of this application is to establish S1PR2-mediated signaling as a key modulator in regulating the immune response to the oral pathogen Aa. We hypothesize that the Aa-induced proinflammatory bone loss response is mediated through S1PR2 signaling. Blocking S1PR2 signaling will reduce proinflammatory cytokine production, attenuate osteoclastogenesis, and alleviate alveolar bone loss induced by Aa. We will test these two specific aims to determine if 1) S1PR2 deficiency in vitro will significantly decrease ERK, PLC, Rho, and NF-?B protein kinase activities, reduce proinflammatory cytokine production, and attenuate osteoclastogenesis in response to Aa exposure. Additionally, we will determine if blocking S1PR2 will decrease osteoclastogenic factors RANK, RANKL, and M-CSF. 2) S1PR2 deficiency in vivo will decrease periodontal leukocyte infiltration, alleviate proinflammatory cytokine production, and attenuate alveolar bone loss. Under the first aim, we will use BMM or preosteoclasts derived from S1pr2+/+ and S1pr2-/- mice to determine if S1PR2 deficiency in cells will reduce the immune response to Aa. Additionally, we will determine if blocking S1PR2 will decrease RANK, RANKL, and M-CSF induced by Aa in BMM or osteoblasts. Under the second aim, we will use an Aa-induced periodontal inflammatory bone loss animal model to determine if S1PR2 deficiency in mice will reduce the inflammatory bone loss response induced by Aa. This study will elucidate the role of S1PR2 signaling, a novel key mechanism, in the pathogenesis of periodontitis. This study will define a new therapeutic target and lay the foundation to develop a novel therapeutic strategy for periodontitis by targeting S1PR2.